inducible responses to dna damage in bacteria and mammalian cells

Environmental and Molecular Mutagenesis 10:9-116 (1987)

Inducible Responses to DNA Damage in Bacteria and Mammalian Cells R.K. Elespuru

Laboratory of Chemical and Physical Carcinogenesis, BRI-Basic Research Program, NCI- Frederick Cancer Research Facility, Frederick, Maryland

Key words: SOS response, mutagenesis, induction

INTRODUCTION

This review summarizes current knowledge of the DNA damage-inducible SOS response in E. coli, with an emphasis on the historical development of experimenta- tion in this area. Evidence for the existence of analogous responses to DNA damage in other bacteria, lower eukaryotes, and mammalian cells is also presented. The mechanistic basis in E. coli for one of the SOS responses, mutagenesis, is currently a subject of intensive study in many laboratories, and recent developments in this area are explored in greater detail. Mutagenesis in mammalian cells, which does not appear to conform to the SOS model, is the subject of a forthcoming review [Rossman and Klein, in press].

For more detailed treatments of various aspects of the SOS response in bacteria and mammalian cells, other recent reviews are recommended: the SOS response in E. coli [Witkin, 1976; Oishi et al, 1981; Little and Mount, 1982; Gottesman, 1984; Walker, 19841; pKMlOl [Strike and Lodwick, 19871; lambda prophage induction [Roberts and Devoret, 1983; Elespuru, 19841; DNA repair [Hanawalt et al, 1979; Defais et al, 1983; Walker, 1985; Walker et al, 19851; the SOS response in mammalian cells [Radman, 1980; Sarasin, 1985; Rossman and Klein, 1985; Lambert and Garrels, 19861.

Received May 6, 1987; revised and accepted May 21, 1987.

Address reprint requests to R.K. Elespuru, BRI-Basic Research Program, NCI-Frederick Cancer Re- search Facility, P.O. Box B, Frederick, MD 21701.

The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

By acceptance of this article, the publisher or recipient acknowledges the right of the US Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

Published 1987 by Alan R. Liss, Inc.

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THE SOS RESPONSE IN E. coli

Observations of apparently unrelated phenomena (virus reactivation, mutagenesis, prophage induction, filamentation) in different genetic backgrounds in E. coli led to a unifying hypothesis called the “SOS response” [Radman, 19751. The “SOS” hypothesis suggested “the existence of a DNA repair system which is normally repressed but which is induced by DNA damage”. Two early concepts were critical to the development of the SOS model. One was that mutagenesis and repair of DNA damage depended on an inducible cellular process [Sedgewick, 1975; Witkin, 19761. This concept developed from the experiments of Weigle and others, which showed that survival and mutagenesis of UV-irradiated lambda phage depended on the irradiation of the host E. coli and was inhibited by the presence of chloramphenicol Weigle, 1953; Defais et al, 19761. The second concept, of coordinate regulation of responses to DNA damage, arose from genetic experiments. Mutations in certain genes (rec and lex) resulted in the abolition of all of the known responses to genetic damage under study (Weigle reactivation, mutagenesis, lambda prophage induction, filamentation) or to their temperature-dependent expression witkin, 1967, 1969; Castellazzi et al, 1972; Ishii and Kondo, 19751. The abolition of “Weigle reactivation” (enhanced survival of UV-treated lambda grown in irradiated hosts) by rec and lex mutations linked the inducible cellular process to that responsible for coordinate regulation [Defais et al, 1971; Radman, 19751.

The basic elements of the SOS model, in terms of the negative regulation of various DNA damage-inducible functions, were proposed by Gudas and Pardee in 1975. The model stated that the l e d gene encoded a repressor of SOS functions; DNA damage led to the induction of RecA, which inactivated Lex. Consistent with the concept of repressor control of SOS functions was the finding that a protease inhibitor prevented the expression of these functions [Meyn et al, 19771.

The SOS hypothesis has been verified and amplified by subsequent experimen- tation (reviewed in Witkin [1976], Little and Mount [1982], and Walker [1984]). RecA protein was isolated and found to cause or aid in the cleavage of lambda and Lex repressors in vitro [Roberts et al, 1978; Little et al, 1980; Horii et al, 19811. (Temperate prophages such as lambda, integrated into host chromosomes, are repressed by their own repressors rather than by LexA repressor [Roberts and Roberts, 1975; Crow1 et al, 1981; Roberts and Devoret, 19831). Specific sequences to which Lex repressor binds were found in the operator region of the genes that it regulates [Brent and Ptashne, 1981; Little and Mount, 1982; Sancar et al, 19821.

A novel method for locating damage-inducible functions in E. coli was published by Kenyon and Walker in 1980. They tested clones of E. coli containing random insertions of a Mu d(luc) phage for the expression of 6-galactosidase in response to a chemical mutagen. Clones expressing the lucZ gene product contained Mu insertions downstream of damage inducible (din) functions. Five din genes were mapped to discrete locations on the E. coli chromosome, some of which were known to code for DNA repair-related functions. At least 17 functions are now known to be part of the SOS regulatory network walker, 19841 (Table I). These din genes encode a variety of DNA repair enzymes and genes affecting DNA synthesis and cell division [Kenyon and Walker, 1980; Walker, 19841. Expression of din genes enhances the survival of the bacteria (or the bacteriophage in the case of bacteria harboring prophages) and should provide a selective advantage in the presence of environmental mutagens.

Inducible Responses to DNA Damage 99

TABLE I. Bacterial SOS Genes

Gene Function

lexA recA

recN uvrA

B C

uvrD umuC

D

sfiA (sulA) himA cle 1. 2 ssb + A din B din D din F ? ?

TUV

9

Repressor of SOS genes Pleiotropic: cleaves SOS repressors, required for mutagenesis,

Recombination recombination, and repair

Excision repair

Helicase II; mismatch repair Mutagenesis

Survival Filamentation Site-specific recombination Colicins Single strand DNA binding protein ? ? ? ? Respiration inhibition Stable DNA replication Alleviation of restriction

Studies of the kinetics of 0-galactosidase synthesis under control of different din genes fused to lac2 showed that certain genes are expressed sooner than others following SOS-inducing treatment [Kenyon and Walker, 1980; Barb6 et al, 19841. S’A and uvrB are expressed early, or after weak inducing treatments. RecA and umuDC are expressed after stronger treatment [Walker, 19841. Lambda repressor is cleaved only after a long lag period, essentially a whole cell-generation time [Elespuru and Yarmolinsky, 1979; Kenyon and Walker, 19801. These differences were attributed to differences in the dissociation constants for Lex repressor binding to different regions of DNA [Brent and Ptashne, 1981; Little and Mount, 19821 and to slower rates of cleavage of lambda repressor [Little, 19831. However, more recent evidence indicates that additional levels of regulation exist for many SOS genes, including lex, umuDC, and colicin E l (cooperative repressor binding at two sites); uvrB and uvrD (repressor-dependent and repressor-independent promoters; h i d (repressor binding influenced by a host factor, summarized in Walker [1984, 19851); lambda (induction influenced by other SOS functions, eg, ssb [Resnick and Sussman, 19821, sfiA (stability regulated) [Mizusawa and Gottesman, 19831; and umuD and mucA (posttran- slational modification by RecA) [Blanco et al, 1986; Marsh and Walker, 19871. The basal and induced levels of expression of different SOS genes can differ by many fold [Kenyon and Walker, 1980; Markham et al, 1984, 1985; Barb6 et al, 1984; Sedge- wick, 1985; Smith, 1985; Llagostera et al, 1986; Peterson et al, 19861.

The overproduction of RecA protein is not sufficient to cause the expression of damage-inducible genes [Uhlin and Clark, 1981; Quillardet et al, 19821. Rather, the critical repressor-clevage activity of RecA is acquired only when it is activated; activation occurs in vitro in the presence of single-stranded DNA and dATP [Craig and Roberts, 19801 and is thought to involve a conformational change in the RecA protein. Some r e d mutants express the activated phenotype constitutively [Kirby et

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al, 1967; Phiziclq and Roberts, 1981; Tessman et al, 19861. Activation in vivo may occur when RecA encounters single-stranded regions of DNA at gaps, stalled replication forks, or regions of DNA undergoing repair. Besides interaction at single- stranded regions in DNA [West et al, 19801, recent evidence also suggests that RecA binds preferentially to double-stranded DNA containing lesions (pyrimidine dimers) [Lu et al, 19861. It is possible that RecA binds to the lesions themselves; however, lesion-induced distortions in the DNA could create adequate single-stranded regions for RecA binding. Another protein, the single-stranded DNA-binding protein (SSB), appears to facilitate the activation of RecA [Baluch et al, 1980; Weinstock and McEntee, 1981; Cohen et al, 19831.

The current SOS model suggests that DNA damage initiates a cascade of events in E. coli, beginning with the activation of RecA protein and the ensuing cleavage of Lex (and lambda) repressors. Inactivation of free repressor and lowering of intracel- lular repressor concentration causes bound repressor to dissociate from the DNA. Previously repressed din genes are then available for transcription. The repair of DNA by the products (eg, repair enzymes) of the induced genes causes a reversal of the cascade of events and the return of the repressed state; a decrease in the level of damaged DNA results in lower levels of activated RecA protein, increasing levels of intact Lex repressor and increasing levels of repression [Little and Mount, 1982; Little, 1983; Gottesman, 1984; Markham et al, 19851. DNA damage ultimately controls the level of expression of SOS functions.

The precise nature of this damage in DNA, the “inducing signal’’ that is responsible for the activation of RecA protein in vivo, is not known. Biochemical, genetic, and chemical evidence suggests that single-stranded or gapped DNA is involved: 1) The most efficient inducers of SOS functions, such as bleomycin, mitomycin C, and UV light, are efficient inducers of DNA strand breaks, either directly or via repair processes [Rupp and Howard-Flanders, 1968; Love et al, 1981; 2) the cleavage of lambda and Lex repressors by RecA protein in vitro requires the addition of single-stranded DNA [Craig and Roberts, 19801; 3) mutant E. coli such as poZA, which are defective in repairing gapped DNA, are more easily induced [Blanco and Pomes, 19771 while mutants defective in enzymes (Rec BC) which generate breaks on UV or chemically damaged templates are less inducible or noninducible [Little and Hanawalt, 1977; Oishi et al, 19811 for SOS functions by some inducing agents. A correlation between prophage induction and the presence of short oligonucleotides led to the suggestion that DNA degradation products were the inducing signal [Smith and Oishi, 1978; Oishi et al, 19811. However, other DNA intermediates have been proposed based on results in a variety of experimental systems. Indirect induction by certain autonomously replicating plasmids has provided evidence [Bailone et al, 19841 that induction is coupled to the generation of single- stranded regions on replicating DNA. Others [Lu et d , 19861 have argued that DNA- containing lesions such as pyrimidine dimers, to which RecA binds, constitute the inducing signal. The differential activation of RecA protein by intact plasmids such as F’ or pBR322 for mutagenic but not repressor cleavage activity has indicated the possible existence of more than one type of inducing signal to activate the different functions of RecA [Ennis et al, 19851. A similar suggestion regarding the generation of different inducing signals followed observations of differential induction responses to chemicals from functions controlled by different repressors [Smith, 19851.


MUTAGENESIS IN E. coli

Mutagenesis by UV radiation and many chemicals is dependent on induction of SOS functions in E. coli and other prokaryotes. The particular SOS genes involved are recA and the umu (for uv mutagenesis) D and C genes [Kato and Shinoura, 1977; Steinborn, 1978; Bagg and Kenyon, 1981; Marsh et al, 19861 which constitute an operon repressed by Lex [Elledge and Walker, 1983; Kitagawa et al, 19851. The normal progression of the replication complex is thought to be blocked by UV- induced dimers, apurinic sites, and carcinogen adducts [Edenberg, 1975; Doniger, 1978; Villani et al, 1978; Schaaper et al, 19831, a situation which appears to be alleviated in SOS-induced bacteria [Villani et al, 19781. An inducible function allowing synthesis to continue past lesions has long been sought. Such bypass synthesis is thought to be accompanied by a decrease in fidelity, originally termed “error-prone” repair [Witkin, 19761.

Bridges and Woodgate [ 19851 have proposed a two-step model for mutagenesis involving 1) misincorporation and 2) bypass synthesis. They observed that delayed photoreversal of UV lesions results in the generation of particular suppressor mutations in urnu strains that are normally nonmutable [Bockrath et al, 19841. This result suggests that umu and proteins are involved in a late-step in mutagenesis, eg, in the resumption of DNA synthesis on stalled replication forks, subsequent to the misincorporation of bases. Photoreactivation of thymine dimers removes the blocking lesions, obviating the requirement for these proteins in the generation of mutations. Umu proteins could be part of a replication complex with altered synthetic capabilities, allowing replication past DNA lesions. The involvement of the repair polymerase Pol 1 [Lackey et al, 19821 or the replicating polymerase, pol III pridges et al, 1976; Villani et al, 1978; Lu et al, 19861 in bypass synthesis has been suggested. Experi- ments in vitro have demonstrated the inactivation of the proofreading E subunit of pol 111 by recA [Lu et al, 19861. However, a loss of fidelity via the E subunit could not account for the UmuDC or RecA-dependent steps in mutagenesis, since mutants lacking a functional E subunit were still nonmutable in umu- and recA- backgrounds Woodgate et al, 19871.

The non-UV mutability of Lex-defective mutants (no functional repressor; all SOS functions turned on) that also lack activated RecA indicates a requirement for activated RecA in addition to its role in derepression of umuDC Planco et al, 1982; Witkin and Kogoma, 1984; Ennis et al, 19851. The presence of a target sequence for interaction in the umuD protein [Perry et al,1985] suggested that Red-mediated cleavage or other processing of this protein may be required. A correspondence between the state of RecA activation and umu (or muc)-mediated mutagenesis provided strong circumstantial evidence for such processing planco et al, 1986; Marsh et al, 19861. In other recent experiments, the expression of SOS functions was inhibited by the presence of excess mucA gene product, a result consistent with a competition for activated RecA between MucA and Lex repressor [Marsh and Walker, 19871.

Blanco et al [1986] have suggested a model for Umu interaction in which Umu proteins bind to lesions in DNA and participate in the inhibition of DNA synthesis; cleavage of Umu by RecA would then alleviate the replication block, allowing by pass synthesis to occur. Indeed, overproduction of UmuD and C under SOS constitu- tive conditions causes a rapid inhibition of DNA synthesis [Marsh and Walker, 19851.

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This model does not postulate an altered DNA polymerase activity, proof for which has remained elusive. It also allows the inclusion of data indicating that pol III is capable of translesion synthesis in the absence of SOS induction [Livneh, 19861.

pKM101

The plasmid pKM101 contains a set of genes, muc (mutagenesis by uv and chemicals) A and B [Shanabruch and Walker, 19801 which are the functional equivalents of umuD and C (reviewed in Strike and Lodwick [1987]). The mucAB genes suppress the phenotype of umuDC mutants [Walker and Dobson, 19791; both sets of genes are under Lex control. It has recently been found that the muc and umu genes share considerable amino acid homology [Perry et al, 19851, and the respective gene products are similar in size (MucA and UmuD - 16,000; MucB and UmuC - 47,000 mw). Although the pair of umu and muc genes share functional similarities, a single member of the pair is not able to substitute for its equivalent, eg, mucA for umuD in E. coli [Perry et al, 19851. This result suggests the possible interaction of the pairs of gene products, perhaps following cleavage of the smaller protein.

Some strains of bacteria, although inducible for DNA repair, lack the umu genes or a functional equivalent. These strains, including Haemophilus injluenzae [Kimball et al, 19771, Neissena gonorrhoeae [Campbell and Yasbin, 19841, and Streptococcus pneumoniae [Gasc et al, 19801, are nonmutable by UV. As pointed out by Campbell and Yasbin, the absence of umu genes coincides with the ability of these species of bacteria to become “competent” for transformation by exogeous DNA. When a plasmid containing functional muc genes was introduced into H. injluenzue, UV mutability was acquired [Balganesh and Setlow, 19851. In contrast to E. coli, it was possible to separate the functional effects of mucA and mucB in Haemophilus: the latter was sufficient to engender UV resistance in rec- bacteria, while both were necessary for UV mutagenesis. The results further indicated the interesting possibility that nonmutable wild-type bacteria may lack only one of the umu-like genes, in this case the smaller mucA equivalent, since wild-type bacteria appeared to have a chromosomal mucB homolog. MucB functioned to suppress the postreplication repair deficiency in rec- bacteria.

Another species appearing to lack a natural set of urnu genes is Salmonella typhimurium. Salmonella strains have a higher rate of spontaneous mutagenesis and become much more sensitive to mutagenesis by UV and many chemical mutagens when the muc-containing plasmid pKM101 is introduced [MacPhee, 1973; Mortel- mans and Stocker, 1976; McCann et al, 19751. The use of Salmonella strains containing pKMlOl is widespread for the testing of chemical mutagens. In addition to increased sensitivity, the presence of the plasmid has a significant effect on the sequence changes generated by some compounds [McCann et al, 19751. Planar aromatic compounds such as ICR-191, benzo(a)pyrene (B(A)P), DMBA, and aflatoxin B1, which are strictly frameshift mutagens in the absence of pKM101, mutate both frameshift and base-substitution strains when pKM101 is present. Many other compounds which are nonmutagenic in the absence of the plasmid generate both base- substitution and frameshift mutations in its presence (furylfuramide and sterigmato- cystin). Still other compounds induce only one or the other type of sequence change that is stimulated by or wholly dependent upon pKM101 (benzyl chloride, acetoxysaf- role, 4-nitroquinoline- l-oxide). These results indicate that pKM 101, presumably be-


cause of mucAB, creates an environment for response to damage that is qualitatively different from that in its absence; moreovoer, the type of premutational lesion influences the response. Consistent with this interpretation is the observation that pKM101 inhibits the generation of tandem duplications in Salmonella, perhaps by the provision of an alternate pathway for processing damage [Hoffmann et al, 19851. Other recent experiments using a mucA mutant altered in the RecA target site confirm and extend these findings [Marsh, et al, 1986; Marsh and Walker, 19871. The mucA mutant had a reduced capacity for RecA interaction, a reduced level of mutagenesis, and a change in the spectrum of mutations following treatment by UV light.

The expanded repertoire of respones to DNA damage observed in the presence of mucAB was not seen in E. coli with intact umu genes; ICR-191 induced primarily frameshift mutations in umu' E. coli [Calos and Miller, 1981; Shinoura et al, 19831 while B(a)P diol epoxide generated primarily base-substitution mutations (frameshift mutations were not directly analyzed but were estimated to be a minor component) [Eisenstadt et al, 1982; Eisenstadt, 19851. Recent results comparing UV mutagenesis in bacterial strains containing either muc or umu genes on a plasmid have suggested a reason for the differences observed: the mucA gene product is a better substrate for RecA interaction than the UmuD protein (Blanco et al, 1986). This, and perhaps the nature of the umu and muc products themselves, resulted in functional differences in the cells in which they were expressed.

These results all support the concept that mutagen specificity is not an absolute quantity but is dependent on the nature of the umu-like genes responsible for processing DNA damage in each cellular system. Although UV light showed the same specificity in both E. coli and mammalian cells [Lebkowski et al, 19851, some chemical carcinogens appeared to exhibit a mutagen specificity in mammalian cells different from that seen in E. coli [Aust et al, 19841.

Damage-Inducible Genes in Other Organisms

Inducible responses analagous to the SOS system have been discovered in other bacteria annd lower eukaryotes, including S. typhimurium [Pang and Walker, 19831 and Proteus mirabilis [Hofmeister and Eitner, 19811. Inducible DNA repair has been observed in (Bacills subtilis [Fields and Yasbin, 19831, Neurospora crassa [Stadler and Moyer, 1981; Baker, 19831, Saccharomyces cerevisiae [Fabre and Roman, 1977; Eckardt et al, 19781, and Ustilago muydis [Leaper et al, 19801, as documented by split-dose effects or cycloheximide inhibition of repair. Six damage-inducible genes were recovered in S. cerevisiae by using the lucZ-gene fusion technique [Ruby and Szostak, 19851.

It is apparent that a diversity of organisms has preserved an SOS system fairly analogous to that in E. coli. In some cases, critical gene products, such as Red-like proteins, have been shown by genetic complementation to be functional in different species [Eitner et al, 1982; Keener et al, 1984; Goldberg and Mekelanos, 19861.

MAMMALIAN SOS FUNCTIONS? Early experiments

The discovery of the SOS system in E. coli and other organisms occurred largely as a consequence of experimental results in different genetic backgrounds.

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Cloning of essential genes and the isolation of additional mutants, particularly encod- ing defective Lex repressors and activated RecA proteases, led to a detailed understanding of the SOS regulatory network in E. coli. The search for mammalian equivalents of DNA damage-inducible functions was hindered by the lack of analogous mutants in mammalian cells. Early experiments sought evidence for the phenomenology of the bacterial SOS system in mammalian cells (reviewed in Hewitt and Meyn [ 19781, Radman [ 19801, Stone-Wolff and Rossman [1981], Witkin [ 19841, Sarasin [1985], and Rossman and Klein [1985]). This included reactivation and mutagenesis of viruses, error-prone repair, induction of resident viruses, and the induction of proteases. Evidence for the “inducibility” of these phenomena (ie, dependencce on new protein synthesis) was sought in “split-dose” experiments or using the inhibitor cycloheximide. Protease inhibitors were used in an attempt to find mammalian analogies for the key role of bacterial RecA protease in the expression of SOS functions [Lytle et al, 1978; Kennedy and Little, 1978; Kinsella and Radman, 19801.

The early experiments with mammalian cells seldom produced results as quan- titatively convincing as those with bacteria. Nevertheless, the inducible reactivation of mammalian viruses was consistently observed. Evidence for the mutagenesis of incoming viral genomes was conflicting and inconclusive, however [Radman, 1980; Bockstahler, 19811. Many of the viruses were reactivated in the mammalian “control” cells which were unirradiated, ie, uninduced. This caused practical difficulties in the detection of significant increases in survival in irradiated hosts. However, as pointed out by Radman, this phenomenon does have an E. coli equivalent, “indirect induction” of SOS functions by certain irradiated autonomously replicating viruses. Alter- natively, cellular functions for proccessing DNA damage might have been expressed constitutively. “Split-dose” experiments purporting to demonstrate inducible DNA repair of the second dose of UV have been criticized [Witkin, 1984; Rossman and Klein, 19851. So have experiments with cycloheximide, used as the mammalian equivalent of the E. coli protein synthesis inhibitor chloramphenicol, because of its effects on a multitude of cellular functions [Rossman and Klein, 19851.

Experiments With SV4* Experiments with small mammalian DNA viruses such as SV40 and polyoma

met with consistent success and continue to be used as probes to study SOS-like functions in mammalian cells [Cornelis et al, 1980; Defais et al, 1983; Sarasin, 19851. SV40 is a small DNA virus which replicates in some cells and transforms others by integrating into host DNA. It has been well characterized genetically [Tooze, 19801. Because SV40 replicates in the nucleus by using host enzymes for replication, it is considered a good model of a single mammalian replicon [Campbell, 19861. Re- sponses to DNA damage have been substantial under a variety of experimental conditions. The mechanisms underlying these responses are unknown but appear to be dependent on T antigen.

Induction of the expression of integrated SV4, genomes has been documented following treatment with a variety of DNA-damaging agents [Kaplan et al, 1975; Lavi and Etkin, 1981; Moore and Coohill, 1983; Dinsart et al, 19851. Another related DNA virus, polyoma, shows a similar response [Lambert et al, 19831. Levels of intact SV40 or SV4, DNA induced by UV or chemical carcinogens were orders of magnitude greater than control levels. These results are reminiscent of results ob-


tained with induction of bacteriophage lambda in E. coli. The location(s) of SV, in clone E [Kaplan et al, 19751 and other inducible cell lines of SV40 are not known, but one or more copy could be downstream of a transcriptional unit responding to DNA damage (analogous to the insertion of lac2 downstream from din genes; Kenyon and Walker [1980]). In order to address this question, Rossman et a1 (in press) have recently examined the location of SV40 insertions in cloned Chinese hamster embryo cells with different capabilities for amplification of viral sequences. Their Southern blot data did not provide evidence for major differences in the location of viral insertions in the highly amplifiable clones.

The indirect induction of SV40 from transformed cells by fusion with irradiated cells has been reported [Nomura and Oishi, 1984; van der Lubbe et al, 19851. However, the latter authors also reported the failure of irradited exogenous calf thymus DNA to induce SV40, while the same treatment did induce a mutator phenotype in an incoming virus [Dinsart et al, 19851. The use of a DNA which has the ability to replicate may be required for indirect induction, as is the case in E. coli; therefore the failure with calf thymus DNA may not be conclusive. On the other hand, the results of Dinsart indicate that separate mechanisms may exist for the induction of SV40 and the generation of untargeted mutations on an incoming viral genome.

The question of the effect of lesions in DNA (specifically pyrimidine dimers) on DNA replication in mammalian cells has been addressed by using SV40 [Sarasin and Hanawalt, 1980; Stacks et al, 1983; Clark and Hanawalt, 1984; Berger and Edenburg 19861. Berger and Edenburg isolated SV, DNA following UV irradiation of infected monkey cells and analyzed the replication intermediates by electron microscopy. They found that bidirectional replication forks generated on UV-damaged DNA were located asymmetrically with respect to the origin. This result indicated that replication forks encountering lesions ( - 7 dimers/molecule distributed randomly) were not able to progress. In earlier experiments, Sarasin and Hanawalt [ 19801 observed a gradient of [3H]-thymidine incorporation after UV irradiation, with the highest concentration near the origin of replication, also indicating a replication block. Synthesis appeared to resume, however, after a delay in which recombinational exchange of dimers occurred (Clark and Hanawalt, 1984). Translesion synthesis in mammalian cells following a transient replication block has been suggested but not yet demonstrated.

Other inducible Functions

Other DNA damage-inducible phenomena have been studied in mammalian cells. These include the induction of proteins such as plasminogen activator and DNA ligase, various types of gene amplification, alterations in gene expression and metab- olism, activation of enzymes and, more recently, changes in the expression of oncogenes.

Plasminogen activator (PA) is a protease which is induced by UV radiation and chemicals in rodent and human embryo fibroblasts [Mishkin and Reich, 19801. The inhibition of its production by protein synthesis inhibitors indicates inducibility by UV. However PA is also induced by tumor promoters such as TPA [Wigler and Weinstein, 19761, apparently more efficiently than by DNA-damaging agents. Al- though PA superficially resembles RecA protein in its properties of UV inducibility

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and protease activity, there is no evidence that it functions in an analogous manner in regulating gene expression.

Other proteins identified as having increased activity or expression as a response to DNA damage are DNA ligase [Mezzina and Nocentini, 1978; Sarasin, 19851, metallothionein [Lieberman et a1 1983; MacArthur et al, 1985; Angel et al, 19861, and H2 antigen (also induced by interferon) [Rahmsdorf et al, 19831. In addition, the enhanced synthesis of eight proteins was detected by two-dimensional gel electropho- resis following treatment of human diploid fibroblasts with UV, mitomycin C, or TPA [Schorpp et al, 19841. These workers found that serum starvation and some cell culture conditions also led to induction of the proteins, while glucocorticoid hormones inhibited the induction. One of the induced proteins, “EPIF,” was excreted from the cells and caused the induction of the same spectrum of proteins in unirradiated cells. This mechanism might serve to cause the coordinate induction of new proteins in a localized population of cells. UV-, and EPIF-mediated induction of proteins was unaffected by protese inhibitors or superoxide dismutse; hence the induction was not mediated by plasminogen activator or “clastogenic factor” [Emerit and Cerutti, 19821.

Gene amplification is a well-documented response of mammalian cells to radiation, carcinogens, and antitumor agents [Stark and Wahl 1984; Schimke, 1984, 1984a; Fuscoe et al, 1983; Tlsty et al, 19841. The best-studied example is the induced resistance to folate inhibitors via amplification of dihydrofolate reductase genes [Tlsty et al, 19841. Amplification of integrated SV4,, sequences also occurs following UV or carcinogen treatment [Lavi and Etkin, 1981; Dinsart et al, 19851. A related phenomenon in E. coli is the amplification of bacteriophage lambda DNA occurring during prophage induction.

Metabolic changes, including alterations in nucleotide pools [Das et al, 19831 and inhibition of DNA methylation Wilson and Jones, 19831, are reported to occur in response to treatment by chemical mutagens. The increased synthesis of poly(ADP- ribose), using NAD+ as substrate, is another documented response to DNA strand breaks and other types of DNA damage. Increased levels of this chromatin-binding factor arise from the activation of the enzyme poly(ADP-ribose)polymerase. Binding of poly(ADP-ribose) to histone and other DNA and RNA binding proteins affects the structure of chromatin and is coincident with changes in the cell cycle and gene expression. Inhibitors of poly(ADP-ribose) polymerase enhance the sensitivity of cells to DNA damage, inhibit DNA repair and have a variety of other effects [Ueda and Hayaishi, 19851. The mechanistic role of poly(ADP-ribose) in cell rgulation is not clear. It has been variously described as a mediator of DNA damage, causing depletion of NAD+ pools and metabolic shut down [Berger and Berger, 19861, or as a signaling element reflecting the availability of NAD’ under different metabolic conditions [Loetscher et al, 19871.

DNA Damage-Induced Alterations in Oncogenes There is, currently, a great deal of interest in the involvement of activated

oncogenes in carcinogenesis. Some primary human tumors and human and animal tumor cell lines contain detectable alterations in the sequence of oncogenes or in the levels of expression of their DNA, RNA, or protein products. At least four different mechanisms have been associated with the activation of oncogenes in tumors: increased or untimely expression (myc, rus, ubl, myb), amplification (myc, rus, abl,


myb), translocation (ubl, myc, mos), and mutagenesis (H-rus, m u ) [Pimentel, 19861. Although DNA-damaging agents can induce all of these changes in gene expressin or gene sequence under various circumstances, direct evidence for activation of oncogenes by UV or chemicals is limited. The assay used to detect DNA sequence alterations in critical genes involves the transfection of NIH 3T3 cells by DNA derived from carcinogen-induced tumors or, in one recent case, DNA treated directly with carcinogens. Transforming genes are detected by the generation of foci of transformed cells. Using this type of method, specific point mutations in the H-rus gene were found to be present in DNA from a variety of carcinogen-induced tumors: from mammary tumors induced in rats by methylnitrosourea (MNU) [Sukumar et al, 1983; Zarbl et al, 19851; from mouse skin tumors induced by P-propiolactone [Garte et al, 19851, 7,12-dimethylbenzanthracene (DMBA) and dibenz(c,h)acridine [Bizub et al, 1986; Quintanilla et al, 19861; from mouse mammary tumors induced by DMBA [Dandekar et a1 19861; and from mouse hepatomas induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1 ‘-hydroxy-2’ ,3’-dehydroestragole [Wiseman et al, 19861. Another oncogene reported to be activated by a point mutation is the m u oncogene activated in rat neuroblastomas induced by ethylnitrosourea [Bargmann et al, 19861. The direct chemical modification of a cloned H-rus gene by N-acetoxy-2- acetylaminofluorene or an isomer of benzo[u]pyrene diol epoxide, or by heat-induced depurination, also led to its activation [Vousden et al, 19861. The generation of the transforming phenotype was accompanied by point mutations in the 12th or 61st codons of the rus gene in each of the cases mentioned above. The sequence changes observed, eg, a G-+A transition in the case of MNU, were consistent with the mutagen specificity expected from each carcinogen.

In a few instances, the amplification of mutated rus sequences also has been observed [Bizub et a1 1986; Quintanilla et al, 19861. The predominance of activated ras mutations detected in these experiments is most likely a consequence of the selection system used, based on NIH 3T3 cell transformation. However activated rus genes account for only a small proportion of the transforming potential of carcinogen- treated cells. In many cases, the transforming genes (or factors) remain unidentified (eg, Garte et al [1985]). It is likely that the development of systems other than the NIH 3T3 cell assay will be necessary to fully explore the question of oncogene activation as a mechanism of tumor induction by chemical carcinogens. A human cell line which may be transformed by oncogenes not detectable in NIH 3T3 cell transfection assays has recently been described [Tainsky et al, 19871. The results so far indicate that oncogene activation is not a new or unique efect of DNA-damaging agents; it occurs by a variety of mechanisms already attributed to DNA-interacting compounds, eg mutagenesis , gene amplification, and genetic rearrangements. The role of DNA damage-inducible functions in oncogene activation will become understandable in the context of these processes

Future Prospects

Mammalian systems are just becoming amenable to the type of studies that have resulted in so much progress in the understanding of the SOS system in E. coli. Until recently, the only source of radiation-sensitive mutants for studies on mechanisms of recovery from DNA damage were patients with genetic diseases such as xeroderma pigmentosum. Improved techniques for the isolation of many different types of mutants are now being used [Collins and Johnson, 1987; Thompson in press].

108 Elespuru

Complementation of mutations in rodent cells by functions on human chromosomes has facilitated the mapping of some DNA repair genes to particular human chromosomes [Hori et al 1983; Thompson et al 1985; Thompson, in press]. At least one human DNA repair gene has been cloned [Westervelt et al, 19841. Methods for studying DNA repair in active versus silent regions of the mammalian genome have been developed [Madhani et al, 19861. Initial results have suggested that transcriptional activity rather than DNA replication, or replication inhibition, may be associated with efficient repair of damage.

There is reason for optimism in the direction of future research in mammalian systems. However recent publications suggest that fundamental problems may exist either in our assumptions concerning mutagenic mechanisms or in the systems being developed to study mutagenic and carcinogenic processes. These potential problems derive from the discrepancy between expected and observed sequence changes found to be responsible for the biological effects of a number of carcinogenic agents [Doniger et al, 1987; Brash et al, 1987; Duesberg, 19871.

CONCLUSION

The verification of the SOS model provided a framework for the explanation of a variety of results in many different mutant strains of E. coli @if, zub, rec, lex, sJi, Lon, tsl, spr) or in lambda lysogens. Results became understandable in terms of the negative regulation of a coordinated network of genes repressed by Lex and induced by activated RecA. A great wealth of new data fit into the framework of the model and led to the resolution of its elements at the molecular level. Complexity gave way to simplicity. Recent results, however have begun to reveal a new order of complexity within the SOS model. The RecA protein is being assigned an ever-increasing number of functions in recombination, repair, and mutagenesis. Moreover, within the model of coordinated induction of many functions is the realization that their expression is at best only loosely coordinated. The kmetics of induction of different functions within a population varies from a few minutes to a whole generation time. Independent regulatory mechanisms exist for many SOS genes, resulting in different levels of expression under both induced and uninduced conditions. Within a single cell, only one or a few functions may be expressed. All of these observations suggest a more complex regulation of individual SOS functions than was originally envisioned.

The key questions related to the nature of the inducing signal(s) and the mechanism of UmuDC- and RecA-mediated mutagenesis should yield to the intensive research effort under way in many laboratories. Answers to these questions may provide a basis for understanding why bacteria and some higher organisms possess SOS systems. Echols [1981] has suggested that inducible mutagenesis provides an evolutionary advantage for survival under adverse environmental conditions.

That higher organisms and mammalian cells respond to DNA damage by the induction of new proteins and new DNA repair activities is now well established. However, whether these functions are strictly analogous to bacterial SOS functions is in doubt. Mammalian cells respond with the synthesis of new proteins not only to DNA-damaging agents, but also to tumor promoters and perturbations in culture conditions, which are essentially nongenotoxic. This response appears to be mediated by an inducible protein which also affects untreated cells, a mechanism that is formally different from that in E. coli. These differences could reflect the fundamental


differences between survival requirements in a population of unicellular organisms vs multicellular organisms.

Bacterial models have provided a useful framework for the design of experiments to probe mechanisms of DNA repair and mutagenesis in mammalian cells. The recent isolation of useful mutants and advances in experimental techniques should allow a more fruitful search for responses to DNA damage in mammalian systems independent of the constraints of bacterial models.

ACKNOWLEDGMENTS

Research sponsored by the National Cancer Institute, DHHS, under contract No. N01-CO-23909 with Bionetics Research.

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